Methods and compositions for the determination of uric acid in blood

ABSTRACT

COLORIMETRIC METHODS AND COMPOSITIONS FOR THE QUANTITATIVE DETERMINATION OF THE URIC ACID CONTENT OF BLOOD BY TREATING SERUM AT AMBIENT TEMPERATURES WITH AN ALKALINE FERRICYANIDE SOLUTION, FOLLOWED BY THE ADDITION OF FERRIC IONS AND A 5-(2-PYRIDYL)-2H-1,4-BENZODI-   AZEPINE OR WATER SOLUBLE SALE THEREOF TO PRODUCE A BRILLIANT PURPLE COLORED SOLUTION WHICH IS THEN QUANTITATIVELY MEASURED BY STANDARD COLORIMETRIC MEANS.

May15,1973 B. KLEIN 7 3,733,777

METHODS AND COMPOSITIONS FOR THE DETERMINATION OF URIC ACID IN BLOODFiled Jan. 1972 2 Sheets-Sheet 2 40 SPECIMENS/HOURQII WASH RATIO) 60SPECIMENS/HOUR (2=| WASH RATIO) 0.6-

o l l l 2 4 s 8 IO l2 I416 mg URIC ACID/I00 ml FIG. 2

L0: we F m p n n 7 f f 0.6-

7 4o 30 so 40 L 0- SPECIMENS/HOUR (2=l WASH RATIO) FIG. 3

United States Patent- 3,733,177 I METHODS AND COMPOSITIONS FOR THEDETERMINATION OF URIC ACID IN BLOOD Bernard Klein, New Hyde Park, N.Y.,assignor to HolImann-La Roche Inc., Nutley, NJ. Filed Jan. 3, 1972, Ser.No. 214,770

Int. Cl. Gllln 31/22 US. Cl. 23-230 B 11 Claims ABSTRACT OF THEDISCLOSURE Colorimetric methods and compositionsfor the quantitativedetermination of the uric acid content of blood by treating blood serumat ambient temperatures with an alkaline ferricyanidesolution, followedby the addition of ferric ions and a -(2-pyridyl)-2H-1,4-benzodiazepineor water soluble salt thereof to produce a brilliant purple coloredsolution which is then quantitatively measured by standard colorimetricmeans.

BACKGROUND OF THE INVENTION The need for an accurate quantitative methodfor determining the uric acid content in blood which utilizes a smallamount of sample, does not require a large degree of technical skill,thus being readily adaptable to clinical use, and is sufficientlyeconomical for mass screening has long been felt. Additionally, it hasbeen considered most desirable that such a method be readily adaptabletoan automated sequential or continuous flow system inorder that a greatmany samples may be processed rapidly and with the highest possibleaccuracy. Thereis a need-for such an automated sequential or continuousflow system which is capable of highly accurate results for the.diagnostic testing of large numbers of persons for the incidence ofabnormal amounts of uric acid in their blood.

Uric acid is normally present in some quantities in the fluids ofmammals, particularly humans, as a result of normal body functions. In agiven warm blooded animal, the uric acid content of body fluids must beheld within carefully prescribed limits in order to preventundesirabl econsequences. v

For example, ingestion of certain purine-containing foods which normallyhave no effect on blood uric acid levels, may unpredictably elevate uricacid blood levels.

3,733,177 Patented May 15, 1973 methods, although improving over theuricase and alkaline phosphotungstate processes, also do not afford thedegree of sensitivity as'well as adaptability to automated procedureswhich are realized with the methods of the present invention.

The diagnostic compositions and methods of the present invention providea reliable, convenient test for the quantitating of uric acid in theblood as well as affording a method whereby the quantitativedetermination may be carried out in a sequential or continuous flowsystem. Further, the diagnostic compositions and methods of the Ipresent invention overcome many of the disadvantages of the prior artmethods of determining uric acid in blood by not requiring a high degreeof laboratory skill and technology, using a small specimen volume, andbeing highly accurate in the clinical situation.

BRIEF SUMMARY OF THE INVENTION In accordance with the invention, a5-(2-pyridyl)-2H- 1,4-benzodiazepine, or a water-soluble salt thereof,preferably in combination with a buffer, is added with an aqueoussolution of ferric chloride to deproteinized serum to which has beentreated with an aqueous alkaline ferricyanide solution, whereby a purplesolution is obtained which can be quantitated as to its uric acidcontent by standard colorimetric means.-

7 DETAILED DESCRIPTION OF THE INVENTION In accordance with the inventiona compound selected from the group consisting of compounds of theformula wherein A is selected from the group consisting of Suchelevation may be indicative of renal insufficiency.

Uric acid, in abnormally high concentrations in the blood, tends tocrystallize out in the body joints causing a very painful inflammatorycondition known as gOutJHighairic acid blood levels are also known to beassociated'with such conditions as uremia and those characterized-bywhiteblood cells,

an excessive destruction of the nuclei'of e.g., leukemia and pneumonia.1 a I It is therefore important to provide a' test for uric acid whichis both simple and accurate and which can be used as an adjunct toroutine screening'operations in clinics and for periodic screening ofpatients in hospitals, nursing homes andthe like.

Prior art" methods of determining uric acidican ibe boardly classifiedas enzymatic, alkaline phosphotungstate and miscellaneous chemicalcolorimetric. The-enzymatic method utilizing the enzyme uricase suifersthe-disadvam tages of requiring a prolonged incubation'gperiod =withverycareful control of the variables-in.theconditions thus requiringhighly skilled laboratory personnel ergpensive equipment and a greatdeal of timeqThe alkaline phosphotungstate method is, disadvantageous inthat ;it

lacks sensitivity which necessitated theuse of arr-excessively largeamount of sample-andhas e hijbitedadyerse side reactions which createturbidity,whichngives false high readings. vThe morerecently developedchemical hydrogen, lower alkyl; and

is selected-from the C=N and 4 R4 0 B is selected from and CH R isselected from the group consisting of halogen, hydrogen,trifluoromethyl, nitro and amino; R is selected from the groupconsisting of and GEN; and R and R where taken together with theirattached nitrogen atom form a radical selected from the group consistingof piperazinyl, lower alkyl substituted plperazinyh. pyrrolidinyl, loweralkyl substituted pyrrolidinyl, piperidinyl and lower'alkyl substitutedpiperidinyl;

.isiloweralkyl; and R, is selected from the group consisting of loweralkyl and hydrogen and water-soluble salts thereof, preferably incombination with a buffer, is added with an aqueous solution of ferricchloride to deproteinized serum which has been treated with an aqueousalkaline ferricyanide solution, whereby a purple solution is obtainedwhich can be quantitated by standard colorimetric means.

Examples of benzodiazepine compounds of Formula I above which areparticularly suitable as the color-forming reagent in the process ofthis invention include the following:

7-bromo-1,3-dihydro-1- [4- (4-methyl-l-piperazinyl)butyl]--(2-pyridyl)-2H-1,4-benzodiazepin-2-one;

7-amino-1,3-dihydro-5-(2-pyridyl)-2H-,1,4-

benzodiazepin-2-one;

1-methyl-1-[3-(7-bromo 5 (2pyridyl)-1,3-dihydro-2- oxo 2H1,4-benzodiazepine-1-yl)propyl]urea Whose preparation is disclosed inUS. Pat. No. 3,464,978, issued Sept. 2, 1969;

7-bromo-1,3,-dihydro-S- (2-pyridyl)-2H 1,4-

benzodiazepine;

7-amino-1,3-dihydro-1-methyl-5-(2-pyridyl)-1H- 1,4-benzodiazepine;

7-bromo-1,3-dihydro-(3-dimethylaminopropyl)-5-(2-pyridyl)-2H-1,4-benzodiazepin-2-one;

7-bromo-1,3-dihydxo-5-(2-pyridyl)-2H-1,4-benzodiazepin- 2-one-4-oxide',

7-bromo-1,3-dihydro-5-(2-pyridyl) -2H-1,4-benzo diazepin-Z-one;

7-bromo-1,3-dihydro-1-(fl-hydroxypropyl)-2-(2-pyridyl)-2H-1,4-benzodiazepin-2-one; and

7-bromo-5-(2-pyridyl) 1,3 dihydro 1 [3-(N-cyanomethylamino)propyl] 2H1,4 benzodiazepin-Z-one whose preparation is also disclosed in U.S. Pat.No. 3,464,978.

The term lower alkyl as used throughout this specification includes bothstraight and branched chain alkyl groups having from 1 to 7 carbon atomssuch as methyl, ethyl, propyl, isopropyl and the like. The term loweralkanoyloxy refers to both straight chain and branched chain aliphaticcarboxylic acid moieties such as acetoxy, propionyloxy, butyryloxy andthe like. The term halo gen includes bromine, chlorine, fluorine andiodine. Also included within the purview of the present invention arethe water soluble acid addition salts of the compounds of Formula Iabove. Any conventional Water soluble acid addition salts of thecompounds of Formula I above may be utilized in the process of thisinvention to quantitatively determine the iron content of aqueoussolutions. Among the acid addition salts which can be utilized inaccordance with this invention, includes salts of compounds of theFormula I with organic or inorganic acids such as hydrochloric acid,hydrobromic acid, nitric acid, sulfuric acid, acetic acid, formic acid,succinic acid, maleic acid, p-toluenesulfonic acid and the like.

The color differentiation with varying concentrations of ferrous ionsproduced by the compound of Formula I above is such that theconcentration of ferrous ions pro duced by the instant diagnosticreagent compositions in situ can easily be determined by standardcolorimetric instruments. Furthermore, the compounds of Formula I arenot sensitive to extraneous sources and therefore are not affected bytrace contaminants. The method of this invention provides a simplecolorimetric means for quantitatively determining the uric acid contentof serum.

In accordance with the present invention, the uric acid content of serumis determined by treating a deproteinized sample of serum with anaqueous alkaline ferricyanide reagent and allowing the mixture to standfor a reasonable time, e.g., about 5 minutes, 'at ambient, i.e. roomtemperatures, thus forming in solution ferrocyanide ions and allantoin.An aqueous solution containing ferric ions in the form of a watersoluble ferric salt and a compound of Formula I is then added whereinferricyanide ions and ferrous ions are produced. The ferrous ions thusproduced react with the compound of Formula I, preferably in thepresence of a buffer, to produce a brillian deep purple color which isread colorimetrically thus affording a rapid, simple quantitativedetermination of the uric acid content of the sample which is ideallysuited for routine diagnostic use.

In accordance with the present invention, the serum sample to betestedis initially treated with a conventional deproteinizing agent. Acidicdeproteinizing agents such as, for example, trichloroacetic acid ortungstic acid, are preferred. The specimen is well mixed with thedeprotein izing agent in a ratio of 1:10 and centrifuged at high speedto obtain a clear supernate. A 1.0 ml. aliquot of the clear supernatecontaining 0.1 ml. of specimen is treated at ambient temperature withabout 2.0 ml. of an aqueous solution of an alkaline ferricyanide reagentand allowed to stand for about 5 minutes. The mixture is then treatedwith about 1.0 ml. of an aqueous solution of a water soluble ferric saltsuch as, for example, ferric chloride and 2.0 ml. of an aqueous solutionof a compound of Formula I. The solutions are mixed and the absorbanceof the violet blue color which develops over about 10 minutes ismeasured at 580 nm. against both a standard uric acid solution similarlytreated and a reagent blank.

The solution containing ferricyanide ions can be made from any watersoluble ferricyanide salt which does not otherwise interfere with thereaction such as, for example, potassium ferricyanide and sodiumferricyanide. Potassium ferricyanide is preferred in the practice of thepresent invention. This reagent may be made in quantity if so desiredand used as needed. The appropriate amount of potassium ferricyanide isdissolved in an aqueous alkaline medium such as, for example, a 2%sodium carbonate solution. The quantity of ferricyanide salt utilized inpreparing the reagent is variable. However, a sufficient quantity mustbe utilized to react with all the uric acid present in the specimen tofurnish a positive indication of elevated uric acid blood levels whenthe diagonstic method of the present invention is being utilized as adiagnostic or a mass screening tool.

Generally, it is preferred that for each 0.1 ml. of serum to be tested,the reagent solution contains from about 0.18 ,umoles to about 1.511110165, most preferably from about 0.35 moles to about 0.7 ,umoles offerricyanide salt. It is preferred that the ferricyanide reagentsolution to be at a pH of from about 9 to about 11. This is readilyaccomplished by the addition thereto of a sufficient amount of an alkalimetal hydroxide or carbonate. Most preferred for this purpose is sodiumcarbonate.

The quantity of ferric ions added to the sampleferricyanide ion mixtureis again variable However, it is preferred to utilize a quantity offerric ion slightly in excess of the molar quantity of ferricyanide ionsadded to the sample. The utilization of such an excess that there willbe sufficient ferric ions present to react with the ferrocyanide ionsgenerated by the initial reaction between the ferricyanide ions and theuric acid in the sample. The ferric ions may be supplied as any watersoluble ferric salt which does not interfere with the diagnosticdetermination such as, for example, ferric chloride, ferric nitrate,ferric sulfate and the like. Of these, ferric chloride is preferred.

The quantity of the compound of Formula I which is addedto theaqueous-reaction mixture is variable. In all instances, however, theremust be a sufiicient quantity of the compound of Formula I present toreact with all of the ferrous ions generated by the reaction between theferric ions and the ferrocyanide ions. This quantity is mostconveniently determined by equating the quantity of the compound ofFormula I with that of the ferric ions to insure the stoichiometry ofthe chelation reaction.

It is preferred to maintain the test medium ata pH of about 4.0 to about5.0, preferably about 4.5. This can most easily be accomplished byadding suitable buffers to'the ferric ion reagent and the reagentcontaining the compound of Formula I. Buffering these reagents alsomakes them stable in aqueous solution when they are made up in quantityfor large scale laboratory testing.

In general, any recognized butler pair suitable for the maintenance ofsuch a pH rangeas described above can be utilized. Preferably, there canbe utilized as a buffer pair a water soluble salt of acetic acid andacetic acid. Of the Water soluble salts of acetic acid sodiu'rri acetateis preferred. However, ammonium acetate, potassium acetate or otherwater soluble salt of acetic acid can be used, if desired. Although thequantities of the buffer pair comprising awater soluble acetic acid saltand acetic acid are variable, the present invention contemplates the useof a sufiicient quantity of the acid component, e.g., acetic acid, toprovide a final test sample having a pH in the range of from about 4.0to about 5.0; preferably 4.5.-By fiinal test sample is meant a solutioncontaining the ferricyanide ions, the ferric ions and thebenz'odiazepine color reagent. In general, there is contemplated thepreparation of a solution of both the ferric ions and the benzodiazepinecolor former which contains per liter about 1.0 mole of a water solublesalt of acetic acid toabout 1.0 to about 2.0 moles of acetic acid.

From the foregoing description it is evident that the compositions ofthe present inventionmay be utilized or handled as prepared aqueousstock solutions, aqueous concentrates or in a dry powder formLIn 'eitherthe con- 'centrate or the power form, sufficient buifering agents areadded to stabilize the compositions when the working dilutions are madeand maintain the pH ofthe reaction mixture at between 4.0 and 5.0,preferably about 4.5.

In another aspect of the. invention, the diagnostic compositions of theinvention may be packaged in a dry state as a diagnostic kit or reagentsystem. In such; a reagent system, the reagents may be packaged inamounts such that stock solutions can be formed therewith which aresuitable for large scale testing either manually'o'r by continuous flowprocedure as contemplated herein; Alternatively, reagent systems maybeprepared which are suitable for single diagnostic determination. Atypical reagent system would also include uric acid from which -anaqueous solution would be prepared to be utilized as a standard for thecolorimetric determinations. The amount of reagents utilized in a givenreagent system may be easily calculated in relation to the specimenbeing tested from the molar quantities given herein. These calculationsare considered to be Well within the purview of a person skilled in theart. A representative reagent system utilizing specific compounds wouldbe as follows.

The above quantities represent sufficientfreag e nts for 1000 tests.ThusfReagents. A. andfiarehd sso vs 1n 2 liters of deionized water and 2liters of a pH 4.5 acetate buffer, respectively. Reagent B is dissolvedin 1 liter of a 1 normal hydrochloric acid. The uric acid standard maybe prepared as a 100ml. stock solution as.described by Caraway in fsta'ndar d Methods of Clinical Chemistry (D. Seligson, eel), v01. Apps,ass-gagAsadsmis Press, NewYork (196 3). This stock: solution canthen beconveniently diluted to form standards for comparative purposes. It isalso within the purview of the invention to prepare a reagent system fora single test utilizing quantities representing one thousandth of thosegiven above. Further, the uric acid standard can be packaged in dry formor as an aqueous solution prepared as above.

In utilizing the compositions of the present invention, the addition ofthe compound of Formula I to the test system immediately produces thedesired purple coloration. The color deepens as the reaction proceeds tocompletion. Accordingly, in order to insure uniform coloring, theaqueous solution should be allowed to stand until its color appears tohave become constant. *In general, it has been found that the fulldevelopment of the purple color will occur over a period of from about 5to 15 minutes after the addition of the compound of Formula I. In mostcases 10 minutes is a sufficient period of time to allow for full colordevelopment.

The quantitation of the uric acid in the colored sample can be carriedout by any conventional colorimetric method utilizing standardspectrophotometers such as Beckman Spectrophotometer, ColemanSpectrophotometer and the like.

The principle of the diagnostic method according to the presentinvention is based on a series of coupled reactions. Initially, uricacid present in the sample undergoing analysis reduces the ferricyanideion in the added first reagent to ferrocyanide ions, which in turn formferricyanide ions and ferrous ions with the addition of the secondreagent which comprises a source of ferric ions such as, for example,ferric chloride, a buffer and a com pound of the Formula I. The ferrousions thus generated react with the compound of the Formula I to producea brilliant deep purple color. The purple color is thereaftercolorimetrically measured and the uric acid content of the samplequantitatively determined.

The quantitative determination of the uric acid content in a specimen iscarried out as follows: the absorbance of the purple color developed inthe sample by the method of the present invention is measured against areagent blank at 580 nm. utilizing a standard spectrophotometer such as,for example, a Beckman DBG Spectrophotometer, employing a cuvette with a10 mm. light path. The quantity of uric acid in the specimen isdetermined in the conventional manner from the absorbance of thespecimen with reference to the absorbance of the color produced by auric acid standard similarly treated. The uric acid content of thespecimen is calculated in accordance with the following formula:

Uric acid content of specimen (mg. ml.)

Uric acid content of standard (mg/100 ml.)

Absorbance of specimen 'Absorbance of standard apparatus. The lattermethod consists essentially of mixing specimens in continuous sequentialflow with normal saline, dialyzing the mixture to produce an aqueousprotein-free solution containing the uric acid, mixing the aqueoussolution with an aqueous alkaline solution containing ferricyanide ions,mixing the aqueous solution with an aqueous solution of a ferric saltand a compound of the Formula I at a constant pH of from about 4.5 toabout 5.5 and passing the resulting solution through an apparatus whichquantitatively determines the glucose content thereof photometrically.

FIG. 1 is a schematic flow diagram illustrating a continuous flowautomated system for analyzing uric acid in biological fluids utilizingthe diagnostic composition of the present invention.

FIG. 2 is a recording of the photometric response obtained whenutilizing the automated system of FIG. 1.

FIG. 3 is a plot in terms of absorbance of the photometric responseillustrated in FIG. 2.

In FIG. 1 a continuous flow automated testing system is shownschematically wherein a specimen sample to be tested, i.e., serum, isdrawn up in sequence from separate sample cups in the sample plate whichrotates at a constant speed to provide the system with 20-60 specimensamples with a 2:1 wash ratio per hour. A sample, so drawn, is mixed inflow with normal saline and passed through a glass mixing coil ofconventional design. After the mixture has passed through the mixingcoil, it is next pumped through a dialyzer module that is provided witha cellophane membrane or the like through which the uric acid passes inaqueous solution by dialysis. The dialyzer module is maintained at aconstant temperature of 37 C. The residual, non-diffusable portion ofthe sample is discarded. As the aqueous uric acid solution passesthrough the dialyzer module membrane it is admixed with an aqueousalkaline solution containing ferricyanide ions, preferably in the formof potassium ferricyanide which react to form allantoin and ferrocyanideions. The aqueous stream is then mixed in continuous flow with anaqueous solution containing ferric ions, preferably in the form offerric chloride, and a reagent stream comprising the 5-(2-pyridyl) 2H1,4- benzodiazepine color reagent of Formula I. The color reagent,preferably 7-bromo 1,3 dihydro-1-(3-dimethylaminopropyl) 5 (Z-pyridyl)2H 1,4 benzodiazepin-Z-one, is maintained at a pH of about 4.5 to 5.5,preferably at about 5.0. The mixture is then passed through a secondmixing coil. As the mixture is in transit through this coil, the ferricions and ferrocyanide ions react to form ferricyanide ions and ferrousions which in turn react with the benzodiazepine color reagent to form abrilliant purple coloration. Photometric measurements are then performedat 580 nm. in a mm. flowcell colorimeter, i.e., the absorbance of thesolution to be tested in measured at 580 nm. in a flow-cell colorimeterusing a 580 nm. filter. The results of the colormetric readings arerecorded on a conventional recording mechanism.

The continuous flow system illustrated in FIG. 1 aspirates at a rate ofto 60 specimens/hour. The rate of flow in ml./min. of the materialsentering the system according to a preferred technique is illustrated inFIG. 1. The materials entering the system are pumped into it by anysuitable pumping means adjusted to maintain the rate of flow illustratedin FIG. 1. The mechanism for the system of the present invention can beconveniently provided by a manifold assembly prepared in accordance withthe system illustrated in FIG. 1 adaptable to the TechniconAutoanalyzer.

In FIG. 2 the absorbance of solutions containing graduated amounts ofuric acid, e.g., 2 rug/100 ml., 4 mg./ 100 ml., 8 mg./ 100 ml., etc. areplotted as a graph against concentration.

In FIG. 3 the photometric response of solutions containing dilferentconcentrations of uric acid is demonstrated. The drawing illustratesfour separate experiments, each of which represents passage through theautomated system of FIG. 1 of a sequence of at least three solutionshaving uric acid concentrations in the order of low to high to low, suchas, for example, 2 mg. per

8 100 ml. to 16 mg. per 100 ml. to 2 mg. per 100 ml. These experimentswere conducted to illustrate the sensitivity of the automated system.The difference in the response curve for similar concentration sequencesrepresents a variance in the speed with which they were passed throughthe system.

The reagents utilized in connection with the automated procedure of uricacid determination comprise aqueous solutions of a ferricyanide reagent,a ferric ion containing reagent and the buffered color forming reagent.The ferricyanide reagent comprises sufiicient ferricyanide to react withall the uric acid in the sample, for example, 0.115 g. potassiumferricyanide dissolved in 1 liter of 2.0% sodium carbonate and 0.9%sodium chloride. The ferric ion containing solutions comprisessufiicient ferric ions to react with all the ferrocyanide ions formed inthe initial reaction, for example, 0.27 g. ferric chloride dissolved in1 liter of 1 N hydrochloric acid. The colorforming reagent comprisessufiicient color-forming compound to react with the ferrous in the ionsformed by the reaction of the ferric ions and the ferrocyanide ions, forexample, 1.88 g; of a compound of Formula I, 82.0 g. of anhydrous sodiumacetate and approximately 40.0 ml. of glacial acetic acid in a liter ofdistilled water. The pH of the solution is maintained between about 4.4and 4.6.

In the practice of the invention according to the automated procedure,iron-free distilled water is pumped through the system for 10 minutes.The system is then switched to reagent and the pumping is continueduntil a steady base line is obtained on the recorder chart. The baseline is set to -0.0lA percent transmission).

The standards in the sample tray are aspirated at a rate of 20 to 60(2:1 wash ratio) samples per hour. The specimens to be analyzed are thensampled, with a standard uric acid specimen which is aspiratedintermittently to insure qualitative control.

The uric acid content of each specimen is determined by reference to acalibration curve prepared by plotting the corrected absorbances of theuric acid standards against concentrations in mg./ ml. Table I setsforth a comparison of results obtained when 10 randomly selected plasmaspecimens were analyzed utilizing the automated and manual uric acidprocedures of the present invention and a prior art method ofdetermining uric acid utilizing uric acid oxidase. The uric acid oxidasemethod utilized was that described by Remp in Standard Methods ofClinical Chemistry (R. P. MacDonald, ed.,) vol. 6, pp. 1-12, AcademicPress, New York, New York 15 75.

,...I n Tabl e II, the recovery of uric acid added to pooled serumaliquots as well as standard uric acid solutions and serum standardcombinations is given. An average recovery of 99.97% (98.7%-l01.0%) wasrealized.

Total uric Found uric L acid, mg./100 acid, 1ng./100 Recovery,

7 ml... m

Specimens combined in 1:1 ratio (mg/100 ml.) I percent Serum Pool #1,6.4 plus Serum Pool #2, 5,5 j 6. 6. 0 100.0 Serum Pool #1, 6.4 plusSerum Pool #3, 6.3 6. 4 6. 5 101. 5 Serum Pool #1, 6.4 plus Serum Pool#4, 7.4 6.9 7.1 Y 102. 8 Serum Pool #2, 5.5 plus Serum Pool #3, 6.3-.5.9 6.0 101. 7 Serum Pool #2, 5.5 plus Serum Pool #4, 7 .4 0. 5 6. 6101. 5 Serum Pool #3, 6.3 plus Serum Pool #4, 7.4 6. 8 6. 7 98. 5

Mean I 101.0

Standard, 2.0 plus Standard, 4.0 3. 0 2. 9 96. 6 Standard, 4.0 plusStandard, 16.0 10. 0 10. 1 101. 0 Standard, 2.0 plus Standard, 16.0 9. 09. 0 100. 0 Standard, 8.0 plus Standard, 16.0 12.0 12. 4 103. 3

Mean. 100.2 Serum Pool #1, 6.4 plus Standard, 2.0 4. 2 "4.1 07.? SerumPool #1, 6. 4 plus Standard, 4.0 5. 2 5. 1 98. 0 Serum Pool #1, 6.4.plus Standard, 8.0 7.2 7.1 98.6 Serum Pool #1, 6.4. plus Standard,12.0.. 9. 2 9. 5 103.2 Serum Pool #1, 6.4 plus Standard, 16.0 11.2 11.2100.0 Serum Pool #1, 6.4. plus water 3. 2 3. 1 96.8 Serum Pool #1, 6.4.plus LiCOa solution 3.2 3.1 96. 8

Mean. 98.7

Composite mean 99. 9

The following examples further illustrate the inven- 2.0 ml. of a colorreagent prepared by dissolving 1.88 tion. All temperatures are indegrees centigrade. g. of7br0m0-l,3-dihydro-1-(3-dimethylaminopropyl)-5- EXAMPLE 1 V (2pyridyl)-2 L I -1,4-benzodiazepin-2-one dihydrochloride I 1 in one literof a pH 4.5 acetate buffer. The acetate buffer To a stirred solutlon of22.0 g. of 7-bromo1,3-d1hydr0- was prepared by dissolving 272.0 g.sodium acetate tri- 5-(2-py11dyl)-2H1,4-benzod1azep1n-2-one 1n. 55.0 ml.of hydrate and 80.0 ml. glacial acetic acid in 500 ml. of dryN,N-dimethylformam1de Was treated wlth 11.0 m deionized water, adjustingthe pH if necessary and dilut- Of a methanolic sOlutiOn of sodiummethoxide (0.0835 ing to one liter, The solutions were, thoroughly mixedmole of NaOCH and stirred for 30 minutes. After 30 and the absorbance ofthe violet blue color that developed minutes, 15.0 ml. of a toluenesolution containing 0.0174 was measured after about 10 minutes against areagent mole of 'y-dimethylaminopropyl chloride 'Was thereafter blank at580 nm. in a Beckman DBG Spectrophotometer added, and the mixturestirred at 75 for 5.5 hours. S0lusinga cuvette with a 10 mm. light path.vents were removed under reduced pressure. and the resi- The uric acidcontent of the specimens was obtained dual oil was dissolved in 100 ml.of dichlorornethane The by reference to a calibration curve prepared byplotting resultant solution Was washed with water, dried and evaptheabsorbances (A) given by standard uric acid solutions orated. The oilwas next dissolved in .100 ml. of ethyl 40 treated in the same manneragainst concentration or by acetate and filtered over 100 g. ofactivated neutral alu-' 1 the Beer-Lambert formula. Utilizing 10 mg.uric acid/100 mina (Grade I); Using ethyl acetate as the eluant, 7- ml.as a standard, the concentration of the specimen was bromo-1,3-dihydro 1(3-dirnethylaminopropyl). 5 (2- calculated according to the formula:pyridyl)-2H-1,4-benzodiazepin-2-one was recovered from the column. 7A580 um. (specimen) EXAMPLE 2 mm (Standard) X10=mg. uric acid/100 ml.The 7-bromo-1,3-dihydro-l-(3 dimethylaminopropyl)- 5 2. 1 4 2 f d in Forcomparatlve purposes, ur c acid analyses were also ample 1 was dissolvedin sufficient methanol to provide conducted on a llke number of Samplesutilizing a a 10 percent solution. This solution was then saturatedbonate'phPsphomngstat-e Procedure described y with hydrogen chloride. Asufficient amount of ether was cafaway 111 Standard Methods of CllnicalChemistry added to cause the solution toturn turbid. The resultantsellgson, L PP. 239-247 Academic Press, New mixture was allowed to coolfor several hours. 7-bromo- York 1,3-dihydro 1(3,-dimethylaminopropyl)-5-(2- id 1)- The results obta ned ut1l1z1ng theaforesaid two tech- 2H-1,4-benzodiazepin-2-one dihydrochlorideprecipitated mques are Set forth In the fQllowlng table! out on standingand 'was separated by filtration. The salt i was recrystallized from amethanol-ether mixture as pale yellow prisms, M.P. 181-183 dec. acid/10Benzo- Pho pho- EXAMPLE V M m 0 s m N dlazepine tungstate Difference,This example demonstrates theapplicability of the test 0 pee en test iDifierence percent vmethod to the uric acid content of blood serum. 2 -30.1 1.6 In the method, 0.5 ml. of serum is deproteinized by the 2:? a?8}, addition of 4.5 ml. of 5% trichloroacetic acid, allowing 1 +011 tifs the mixture to stand at least minutes and centrifuged at 2:8 2:3 is;high speed, e.g., 2,500 r.p.m. to obtain a clear, super- 7-0. 6.8 +012+2I9 natant fluid. A 1.0 ml. aliquot of the clear supernatant 3 3 $1 3582, was treated with 2.0 ml. of a ferricyanide solution .which 9.1 1+011 1 :1 had been prepared by dissolving 0.115 g. of potassium 1 2 18%i' ferric anide in one liter of 27 b ht 6:4 y a 0 y werg aqueous 1 6.0+0.4 +6.3 solution of sodium carbonate and allowed to stand at f? 3:3 g8f g-Z 13% room temperature for about 5 minutes. The mixture was -3 i;713-, f 030 then treated sequentially with 1.0 ml. of a ferric chloride2:8 2'; $8} solution prepared by. dissolving 0.27.0..g: ferric chloride-11 :9 +01 +20 5.6 -0. 2 3.7

hexahydrate in one liter; of 1: N, hydrochloric acid and 11 EXAMPLE 4 Inan analogous manner to that employed in Example 3, tests were conductedwherein uric acid was added to aliquots of serum pools. Over a 20-foldrange of uric acid additions, a mean recovery of 99.5% (range 93.7 to105.7%) was realized. The results of these tests are summarized in thefollowing table.

TABLE Recovery of uric acid added to serum pools Total uric Uric acidpresent; Uric acid acid, g, Uric acid Recovery, in sample, pg. added,pg. calculated found, pg. percent What is claimed is:

wherein A is selected from the group consisting of -C=N and -C=N- B isselected from the group of o H C and CH R is selected from the groupconsisting of halogen, hydrogen, trifluoromethyl, nitro and amino; R isselected from the group consisting of H -R1(|]-R5 hydrogen, lower alkyland 'CI1H211N/ n is an integer from 2 to 7; R is selected from the groupconsisting of hydrogen, hydroxy, lower alkyl,

lower alkoxy and lower alkanoyloxy; R is 2-pyridyl; R is selected fromthe group consisting of lower alkyl, hydrogen,

12 substituted piperidinyl; R is lower alkyl; and R is selected from thegroup consisting of lower alkyl and hydrogen, and water soluble acidaddition salts thereof; and

(d) colorimetrically quantitating the uric acid present by means of saidcolor.

2. The process in accordance with claim 1 wherein said source of ferricions consists essentially of an aqueous solution of ferric chloridebuffered to a pH of between from about 4.0 to about 5.0.

3. The process in accordance with claim 1 wherein said source of ferriciron ions is an aqueous solution of ferric chloride and said source offerricyanide ions is potassium ferricyanide.

4. The process in accordance with claim 1 wherein said color-formingbenzodiazepine compound is added as an aqueous solution buffered to a pHof from about 4.0 to about 5.0 with a buffer pair comprising awatersoluble salt of acetic acid and acetic acid.

5. The process in accordance with claim 4 wherein said color-formingbenzodiazepine compound is selected from the group consisting of7-bromo-1,3-dihydro-l-(3-dimethylaminopfopyl) 5(Z-pyridyl)-2H-l,4-benzodiazepin-Z-one and water soluble acid additionsalts thereof.

6. A method for the quantitative analysis of the uric acid content ofblood serum consisting esesntially of providing in continuous flow thesequential steps comprising:

(a) combining in continuous flow a measured specimen of plasma with anisotonic solution of sodium chloride;

(b) passing said mixture through a dialysis zone, thereby separatingfrom said mixture a clear aqueous solution;

(c) mixing said clear aqueous solution with a measured amount of analkaline aqueous solution of a water-soluble ferricyanide salt;

((1) mixing the aqueous solution produced in step (c) by concurrent flowwith a first reagent comprising a measured amount of a buffered aqueoussolution of a ferric iron salt and a second reagent comprising abuffered, aqueous solution of a color-forming benzodiazepine compoundselected from the group of compounds of the formula wherein A isselected from the group consisting of -C=N and C=N l 4 l 0 B is selectedfrom the group consisting of and CH R is selected from the groupconsisting of halogen, hydrogen, trifluoromethyl, nitro, and amino; R isselected from the group consisting of H R72JRg hydrogen, lower alkyl andCnH2I1N n is an integer from 2 to 7; R is selected from the groupconsisting of hydrogen, hydroxy, lower alkyl, lower alkoxy and loweralkanoyloxy; R is 2-pyridyl;

R is selected from the group consisting of lower alkyl; hydrogen,

and CEN; and R and R where taken together with their attached nitrogenatom, form a radical selected from the group consisting of piperazinyl,lower alkyl substituted piperazinyl, pyrolidinyl, lower alkylsubstituted pyrrolidinyl, piperidinyl, and lower alkyl substitutedpiperidinyl; R is lower alkyl; and R is selected from the groupconsisting of lower alkyl and hydrogen thereof and water soluble acidaddition salts thereof thereby forming a colored so lution; and

(e) flowing said colored solution to an analyzing zone andphotometrically determining quantitatively during the flow of saidcolored solution through said analyzing zone the concentration of uricacid present in said sample.

7. The method in accordance with claim 6 wherein said first reagent andsaid second reagent are buffered to a pH of from about 4.0 to about 5.0with a bulfer pair comprising a Water-soluble salt of acetic acid andacetic acid.

8. The method in accordance with claim 6 wherein said color-formingbenzodiazepine compound is selected from the group consisting of7-bron1o-1,3-dihydro-l-(3-dimethylaminopropyl)-5-(2-pyridyl) 2H 1,4benzodiazepin- 2-one and water-soluble acid addition salts thereof.

9. The method in accordance with claim 6 wherein said water-solubleferricyanide salt is potassium ferricyanide and said Water-solubleferric iron salt is ferric chloride.

10. A reagent system for the determination of the uric acid content ofblood serum samples consisting essentially of:

(a) a first container containing for each 0.1 ml. of serum to be testedfrom about 0.18 to about 1.5 moles of a water-soluble ferricyanide and asufficient amount of an alkalinizing substance selected from the groupconsisting of alkali metal hydroxide and carbonate to render the pH ofan aqueous solution thereof between from about 9 and 11;

(b) a second container containing at least an equimolar amount of awater-soluble ferric iron salt based on the moles of ferricyanidepresent in said reagent (a);

(c) a third container containing at least an equimolar amount of acolor-forming compound selected from the group of compounds representedby the formula wherein A is selected from the group consisting of B isselected from the group of and CH R is selected from the groupconsisting of halogen, hydrogen, trifluoromethyl, nitro and amino; R isselected from the group consisting of H -R7-Ru hydrogen, lower alkyl andR5 -Cu'lIznN 12 is an integer from 2 to 7; R is selected from the groupconsisting of hydrogen, hydroxy, lower alkyl, lower alkoxy and loweralkanoyloxy; R is Z-pyridyl;

R is selected from the group consisting of lower alkyl, hydrogen,

and -CEN; and R and R where taken together with their attached nitrogenatom, form a radical selected from the group consisting of piperazinyl,lower alkyl substituted piperazinyl, pyrrolidinyl, lower alkylsubstituted pyrrolidinyl, piperidinyl and lower alkyl substitutedpiperidinyl; R is lower alkyl; and R is selected from the groupconsisting of lower alkyl and hydrogen, and water soluble acid additionsalts thereof based on the amount of said water-soluble ferric iron saltpresent in reagent (b); and

(d) a fourth container containing, as a standard, a substance selectedfrom the group consisting of uric acid and aqueous solutions thereof.

11. A reagent system in accordance with claim 10 wherein saidwater-soluble ferricyanide salt in said first container is potassiumferricyanide, salt alkalinizing substance is sodium carbonate, saidwater-soluble ferric iron salt in said second container is ferricchloride, said colorforming compound in said third container is selectedfrom the group consisting of7-bromo-1,3-dihydro-1-(3-dimethylaminopropyl) 5(2-pyridyl)-2H-1,4-benzodiazepin-2- one and water-soluble acid additionsalts thereof and said first container contains from about 0.35 to about0.7a moles of potassium ferricyanide.

References Cited UNITED STATES PATENTS 3,449,081 6/1969 Hughes 23---253R 3,506,404 4/1970 Evans et a1. 260-239.3 D X MORRIS O. WOLK, PrimaryExaminer R. M. REESE, Assistant Examiner US. Cl. X.R.

260-2393 D, 239 BD

